PROJECT SUMMARY Highly active antiretroviral therapy has reduced morbidity and mortality from HIV/AIDS but does not lead to a cure. The persistence of the virus within latent reservoirs even in well-treated individuals results in a lifelong commitment to these drug regimens. As a consequence, patients remain burdened by co-morbidities and exposed to the negative social issues that come with being HIV-positive. Therefore, there is an urgent need for the development of therapeutic strategies capable of eradicating virus from individuals, which would greatly improve the lives of people living with HIV/AIDS. In response to this need, we propose to combine the actions of latency reversing agents (LRAs), broadly neutralizing antibodies (bnAbs), and activated natural killer (NK) cells within a single therapeutic platform. More specifically, we propose to use bioengineering strategies to design an ensemble biohybrid therapeutic wherein LRAs and bnAbs are packaged within poly(lactic-co-glycolic acid) (PLGA) nanoparticle depots (nanodepots) and then attached on to the surface of NK cells. We anticipate that the co-localization of LRAs with NK cell activation and local bnAb presence will trigger a more effective and coordinated eradication of persistent HIV reservoirs. In the R21 phase of this project, we propose to engineer NK-nanodepots encapsulating LRAs and bnAbs as off-the-shelf biohybrids that retain the phenotype and function of the constituent NK cells and encapsulated LRAs and bnAbs. The realization of a functional ensemble biohybrid therapeutic that successfully coordinates the actions of LRAs, bnAbs, and NK cells will enable us to proceed to the R33 phase of the project where we will test the efficacy of the ensemble biohybrid therapeutic in eradicating the HIV reservoir in vitro and in a humanized mouse model. Successful completion of this phase of the project will result in a novel therapeutic platform for eradicating persistent HIV reservoirs and will open the way for the translation of our platform to the design and conduct of future clinical trials.